The present invention relates generally to the field of xcex11,3-fucosyltransferases and, more specifically, to xcex11,3-fucosyltransferase polypeptides which are transmembrane segment-free.
Helicobacter pylori is an important human pathogen which causes both gastric and duodenal ulcers and has also been associated with gastric cancer and lymphoma. This microorganism has been shown to express cell surface glycoconjugates including Lewis X, Lewis Y. and sialyl Lewis X. These bacterial oligosaccharides are structurally similar to tumor-associated carbohydrate antigens found in mammals.
The presence of H. pylori isolate has been associated with an increased risk for development of gastric cancer (Wirth, H.-P., Yang, M., Karita, M., and Blaser, M. J. (1996) Infect. Immun. 64 4598-4605). This pathogen is highly adapted to colonize human gastric mucosa and may remain in the stomach with or without causing symptoms for many years. Although H. pylori elicits local as well as systemic antibody responses, it escapes elimination by the host immune response due to its sequestered habitation within human gastric mucosa. Another mechanism by which H. pylori may protect itself from the action of the host immune response is the production of surface antigens mimicking those in the host.
In mammalian cells the enzyme xcex1(1,3/1, 4)-fucosyltransferase (namely FucT) catalyzes the last step in the synthesis of two carbohydrate structures, Galxcex2 1-4[Fucxcex11-3] GlcNAc (Lewis X, Lex for short) or NeuAcxcex12-3-Galxcex2 1-4[Fucxcex11-3]GlcNAc (sialyl Lewis X, sLex for short). (Lowe et al., 1990, Cell 57: 475-484; Kukowska-Latallo et al., 1990, Genes and Development 4:1288-1303.) Cell surface xcex1(1,3)- and xcex1(1,2)-fucosylated oligosaccharides, that is, Lewis X (Lex), sialyl Lewis X (sLex) and Lewis Y (Ley), are present on both eukaryotic and microbial cell surfaces. In mammals, Lex is a stage-specific embryonic antigen, however, Lex, sLex and Ley are also regarded as tumor-associated markers. The biological functions of these bacterial oligosaccharide structures are not fully understood. It has been suggested that such glycoconjugates produced by H. pylori, may mimic host cell antigens and could mask the bacterium from the host immune response. It is also possible that these bacterial Lewis antigens could down regulate the host T-cell response. Therefore, production of such antigens may contribute to colonization and long-term infection of the stomach by H. pylori. 
Presently, use of carbohydrates as potential therapeutic drugs has become popular in the field of medical chemistry. In addition, qualitative and quantitative carbohydrates including Lex, Ley and sLex are also required as reagents for assaying the enzymes which are involved in the biosynthesis of glycoconjugates in cells. Lex, Ley and sLex products which are commercially available are chemically synthesized. However, synthesis of these products gives rise to several limitations such as time-consuming, complicated procedures and low yields. Although several mammalian fucosyltransferases have been cloned and expressed, enzymatic synthesis of Lex, Ley and sLex products for a commercial purpose has not been reported.
The present invention is based on the discovery of a novel xcex11,3-fucosyltransferase polypeptide and gene which encodes the polypeptide. The present invention includes a novel nucleic acid sequence of xcex11,3-fucosyltransferase polypeptide which is useful in the detection and synthesis of xcex11,3-fucosyltransferase polypeptide.
In another embodiment, the invention provides a method of using the novel xcex11,3-fucosyltransferase to synthesize oligosaccharides such as Lex, Ley and sLex.
In another embodiment the invention provides the novel polypeptide of xcex11,3-fucosyltransferase which is useful in the development of antibodies to xcex11,3-fucosyltransferase.
In another embodiment, the novel polypeptide of xcex11,3-fucosyltransferase has a carboxyl terminal xcx9c100 amino acids in length having therein a heptad repeat of X1X2LRX3X4Y, wherein X1 is D or N; X2 is D or N; X3 is I, V or A; X4 is N or D. In another embodiment, the xcex11,3-fucosyltransferase is a peptide selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In another embodiment the xcex11,3-fucosyltransferase may have a variable number of heptad repeats.
Further provided is a method for producing xcex11,3-fucosyltransferase. The method involves the step of culturing a gene expression system which comprises a host cell which has been recombinantly modified with a polynucleotide encoding xcex11,3-fucosyltransferase or a portion thereof and harvesting the xcex11,3-fucosyltransferase. A preferred embodiment of the method is directed to the use of the claimed genetic expression system which produces xcex11,3-fucosyltransferase.
These and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the Examples.
The abbreviation used are: FucT, xcex11,3-fucosyltransferase unless specified otherwise; Lex, Lewis X; sLex, sialyl-Lewis X; Ley, Lewis Y; nt, nucleotide (s); kb, kilobase (s); aa, amino acid (s); PCR, polymerase chain reaction; ORF, open reading frame; RSB, a ribosomal binding site; LPS, lipopolysaccharides; HD-Zip, homeodomain-leucine zipper; bZip, basic region-zipper; LacNAc-R, Galxcex21-4GlcNAcxcex2-Oxe2x80x94(CH2),COOMe; Galxcex21-3GlcNAc-R, Galxcex21-3GlcNacb-Oxe2x80x94(CH2)8COOMe; LacNAc-TMR, Galxcex21-4GlcNAcxcex2-Oxe2x80x94(CH2)8COxe2x80x94NHCH2CH2NH-TMR; Phenyl-Gal, phenyl-xcex2-galactoside.